常见食源性感染致病菌属、种用PCR-RFLP及PCR-SSCP技术鉴定探讨

Application of PCR-RFLP and PCR-SSCP Techniques in the Genus/Species Discriminative Diagnosis of Foodborne Pathogenic Bacteria

【目的】探讨运用聚合酶链反应邛艮制性片段长度多态性分析(PCR-RFLP)和聚合酶链反应-单链构象多态性分析(PCR-SSCP)进行食源性感染常见致病菌属种鉴定的可行性。【方法】选择细菌23S rDNA基因作为鉴别细菌属的靶基因,通过设计合适的通用引物进行PCR扩增,并对13属食源性感染常见致病菌和5种不同血清型大肠杆菌扩增产物的酶切片段进行RFLP分析和SSCP分析。【结果】运用通用引物可以扩增出13 属食源性感染常见致病菌的23S rDNA基因HpaⅡ酶切产物的RFLP分析表明,不同种属的致病菌表现出不同的RFLP图谱,利于进行细菌属的鉴定。不同种属食源性感染常见致病菌SSCP图谱变异性更大,不利于进行种属间鉴别。对5种不同血清型大肠杆菌的23S rDNA基因扩增产物的RFLP和SSCP分析表明,不同血清型大肠杆菌表现出相似的RFLP图谱。SSCP图谱的变异性较大,这有利于进行血清型间鉴别甚至株间的鉴别。【结论】PCR-RFLP和PCR-SSCP具有应用于食源性感染常见致病菌种、属鉴别诊断的潜力。

[Objective]To study the feasibility of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR-single strand conformation polymorphism (PCR-SSCP) technology in the genus/ species discriminative diagnosis of foodborne pathogenic bacteria. [Methods] 23S rDNA gene was selected as target fragment and universal oligonucleotide primers were designed and synthesized. PCR products of 13 genra of foodborne pathogenic bacteria and 5 serotypes E.coli were digested with restriction enzyme Hpa Ⅱ, and analyzed with RFLP and SSCP. [Results] 23S rDNA gene fragment could be amplified from all the 13 genra of foodborne pathogenic bacteria. RFLP analysis of enzyme product manifested that different genra bacteria show different RFLP enzymogram, which are faciliated to genra discrimination. With respect to SSCP analysis, much more mutate fragment displayed in different genra of foodbome pathogenic bacteria was not benefit to genra discrimination. RFLP analysis of enzymplysis product amplified from 5 serotypes E.coli manifested that different serotype E.coli display similar RFLP enzymogram. With respect to SSCP analysis, much more mutate fragment displayed in different serotype E.coli might be benefit to serotype or strain discrimination. [Conclusion] PCR-RFLP and PCR-SSCP are useful in the genra/species discrimination diagnosis of foodborne pathogenic bacteria.